Retroviruses have the capacity to infect a wide variety of cells. Beside this, retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript. This genomic RNA is after infection and reverse transcription integrated into the DNA genome of the target cell. For propagation of infectious virus all replication-competent retroviruses encode as essential genes the so-called gag, pol and env genes that are transcribed from the transcription-regulatory elements contained in the U3-region of the 5′LTR. This transcription starts at the border of the U3- to the R-region of the 5′LTR and the mature transcript finishes at the polyadenylation site at the end of the R-region in the 3′LTR. The resulting RNA transcripts comprise full-length as well as spliced retroviral RNA. The 5′-end of the full-length as well as of the spliced retroviral RNA is modified by addition of a so-called capping group. This structure is important for the attachment of ribosomes and thereby for the translation of the RNA. Translation requires besides this binding signal for a ribosome a so-called open-reading-frame ORF, i.e. a DNA or RNA sequence between an ATG/AUG translation start signal and a termination codon. In normal retroviruses RNA transcripts comprise only one ORF (so-called monocistronic RNA). This monocistronic RNA is capped and translation of the ORF starts at the first translation-start-codon (e.g. ATG) following the capping group and stops at a stop-codon. Consequently, any coding region downstream of said stop-codon wont be translated into a protein. An example for a spliced and capped RNA transcript coding for a single protein is the RNA coding for env. Other essential retroviral proteins, such as e.g. the integrase, reverse transcriptase, protease and capsid protein may be translated as one polypeptide from the capped, full-length RNA transcript. After translation, this polypeptide is proteotytically processed to the different proteins. Hence, this RNA is still monocistronic.
In further developments retroviral vectors have been constructed, which comprise a cassette consisting of a translational control element preceding a heterologous gene (44; 3; 45; 26). In these cases, translation of the one ORF, which is closest to the capping group, starts—as described above—at the first translation-start-codon (ATG or AUG) following the capping group and stops at a stop-codon. For the translation of any further ORF encoded by such a retroviral RNA transcript an additional translational control element, e.g. an internal ribosome entry site (IRES) is necessary.
The term “internal ribosome entry site” (IRES) defines a sequence motive which promotes attachment of ribosomes to that motive on internal mRNA sequences. Furthermore, all factors needed to efficiently start translation at the AUG-start-codon following said IRES attach to this sequence motive. Consequently, an mRNA containing a sequence motive of a translation control element, e.g. IRES, results in two translational products, one initiating from the 5′end of the mRNA and the other by an internal translation mechanism mediated by IRES.
Accordingly, the insertion of a translational control element, such as IRES, operably linked to an ORF into a retroviral genome allows the translation of this additional ORF from a viral RNA transcript. Such RNA transcripts with the capacity to allow translation of two or more ORF are designated bi- or polycistronic RNA transcripts, respectively.
A retroviral vector is characterised by the ability to harbour a heterologous nucleotide sequence in addition to the vector sequence and to transfer said sequence into a receipient. However, for the following reasons, the replication competence of the retrovirus is often lost when a nucleotide sequence is added into the vector. Most retroviruses are adapted in a way that they contain as little RNA as possible and, therefore, contain only essential genes. This is especially true for the simple retroviruses, such as MMTV and MLV, which basically contain only genes encoding virion proteins. Accordingly, insertion of a heterologous sequence into any gene and thereby the inactivation of said viral gene, results in the loss of the replication competence. Additionally, as described above, the RNA-transcripts mostly encode more than one protein, wherein the nucleotide sequences coding for the different proteins sometimes overlap. Hence, the heterologous sequence can also not be added in between of two genes without destroying a coding region. Furthermore, it is known that the nucleotide sequence, which can be efficiently replicated by the retroviral replication machinery, is highly limited in its length, i.e., regularly genes of the viral genome have to be deleted, to have “enough space” for the heterologous sequence, that is added. The deletion of viral sequences again results in the loss of the replication competence. Finally, the insertion of a sequence, especially of sequences that regulate transcription, such as a promoter, or splice donor and acceptor sites often results in regulatory problems. Retroviruses utilise for all processes of transcription, RNA processing and translation several host cell mechanisms. Accordingly, various cis-acting sequences, either located in coding or in non-coding regions have been described for different retroviral genomes. These cis-acting sequences interact with various host cell proteins to regulate gene expression, RNA processing (15), polyadenylation (24), stability (46; 47), or nuclear export of viral RNA (48). Accordingly, it must be expected that the disruption of any such cis-acting elements severely impairs viral replication and productive generation of infectious viral particles, respectively. This is in line with a report by Yin & Hu who found that insertion of a translational cassette into the viral genome can severely influence or destroy viral propagation (45). Yin & Hu showed that the insertion of a cassette containing an IRES attached to a heterologous gene between the LTR and env-coding sequence of the viral body can—probably due to splicing interference (45)—destroy particle production of the used retroviral vector. Accordingly, this region or at least parts of this region are essential for viral replication and very sensitive to alterations.
Furthermore, complications of viral replication capacity, probably due to disrupted cis-acting sequences, aberrant transcripts or promoter interference, have been reported for recombinant retroviruses or retroviral vectors carrying an expression cassette with an SV40 promoter followed by dihydrofolate reductase gene (dhfr) (36, 37) in the 5′-end of the retroviral LTR.
Accordingly, all previously reported retroviral vectors carrying an IRES cassette have lost the ability to replicate in normal cells. Only Murakami et al. reported an avian retroviral vector construct comprising an IRES translational-cassette at a site at which the oncogene src has been deleted, which retained replication-competence for a few passages. However, said vector showed reduced expression levels of the heterologous gene (26). Since the virus used was an avian retrovirus it is not replication competent in mammalian cells.
As yet, it is not completely understood how cis-acting elements influence or control the viral life cycle. Nevertheless, it seems to be clear that disruption of cis-acting elements by randomly inserting a cassette into the genome of recombinant retroviruses results in promoter interference (9), disturbed splicing balance (46) or lack in packaging efficiency (8), and finally leads to the loss of viral replication or decreasing viral titers.